Module 2
Safety precautions for
AFB culture and DST of
M. tuberculosis
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Learning objectives
At the end of this module, you will be able to:
explain airborne transmission of TB;
adhere to safe practice;
explain the biosafety levels of laboratories for TB
activities;
work safely in biological safety cabinets;
use adequate protective personal equipment;
use appropriate disinfectants;
cope with accidents in the TB laboratory;
handle chemicals safely in the laboratory .
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Content outline
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Transmission of TB
Biohazards in a TB laboratory
Minimum WHO recommendations for TB culture / DST
facilities
Standard safe practice in a TB laboratory
Personal protective equipment
Disinfectants active against M. tuberculosis
Safe disposal of infectious waste
Coping with accidents
Chemical safety
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Transmission of tuberculosis
Mycobacterium tuberculosis is almost always
transmitted by patients with active pulmonary disease:
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TB patient expels bacilli in small droplets of respiratory
secretions.
Secretions quickly evaporate leaving “droplet nuclei” less
than 5 μm in diameter.
Droplet nuclei of this size, containing 1–3 bacilli, can remain
suspended in the air.
Following inhalation, droplet nuclei are able to reach deep
into the lungs to produce infection.
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Aerosol formation: spread of droplets
Coughing
Sneezing
Talking
Singing
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Specimens must be collected
outside the laboratory
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Biohazards in the laboratory: be aware!
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Inhalation hazards: handling of liquids containing TB bacill
generates infectious aerosols:
– pipetting
– working with loops
– centrifugation
– opening tubes
– vortexing suspensions
Ingestion hazards
Inoculation hazards
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Importance of laboratory safety
for preventing
laboratory-acquired infection
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Conclusion of the survey:
•DST is associated with the higher risk.
•Smear microscopy alone does not pose a higher
risk than clerical work.
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Medical fitness of laboratory staff
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In accordance with national laws and practices, health
surveillance of TB laboratory workers should be performed:
– before enrolment in the TB laboratory;
– at regular intervals thereafter;
– after any biohazard incident.
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Workers should be educated about the symptoms of TB and
provided with ready access to free medical care if symptoms
arise.
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Confidential HIV counselling and testing should be offered.
Reassignment of HIV-positive workers away from high-risk
environments should be considered.
[to be adjusted to local policy]
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Biological safety cabinet
The biological safety cabinet, or
BSC, is the single most important
equipment for containing and
quickly eliminating infectious
aerosols generated during
culture/DST procedures.
For proper functioning, the BSC
must be well maintained.
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HEPA filter
• HEPA = high-efficiency particulate air.
• Traps and removes 99.97% of airborne particles equal to
or larger than 0.3 µm in diameter.
• Evaluation of the performance of the HEPA filter and
regular maintenance of the BSC are critical for protection
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Work in a BSC
BSC work zone should be
divided into three areas to
minimize contamination
over items:
– a clean area: supplies;
– working area: specimen;
– contaminated area:
waste container.
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Work in a BSC
• Organize a realistic workload in the BSC. Do not
overload, e.g. no more than 6–8 specimens for
processing at a time, according to centrifuge
capacity.
• All needed material should be present in the
BSC so that work is not interrupted and moves
in and out of the BSC are minimized.
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Is flame allowed in the BSC?
• Avoid continuous flame =
permanent source of heat
• Use intermittent burner
• Can be used in BSC I or II
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Biosafety levels
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WHO recommendations for BSL2
(specimen processing for TB cultures)
• BSCs should be ducted or vented to the
outside.
• Air from BSCs must not be allowed to
recirculate into the room in TB laboratories.
• An adequate budget for regular maintenance
and servicing is essential.
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WHO recommendations for handling TB
cultures
In addition to BSL2 recommendations:
• Isolation of the laboratory (containment room).
• Anteroom with a double-door entry; may consist of
a BSL2 room
• Ventilation: directional airflow, controlled
ventilating system.
• Autoclave on site (but not mandatory in the
containment room).
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Laboratory for handling TB cultures
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Which biosafety level for
TB laboratory activities?
Microscopy in a well-ventilated room
Culture = specimen processing in a BSC in a BSL2
Culture identification and DST
= BSL2 containment room with ventilation:
directional airflow from functionally clean to dirty areas,
controlled ventilating system ensuring 6 to 12 air
exchanges per hour.
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Standard practices
• Limited access to the laboratory.
• No eating, drinking, smoking, etc.
• No mouth-pipetting, no chewing
pencils, etc.
• Assume that ALL specimens are
potentially infectious.
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Standard practices
• Hand-washing (dry with
disposable paper).
• Work surfaces to be
decontaminated at least
once a day.
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Good microbiological techniques
• Good microbiological techniques (GMT): working
methods applied to minimize exposure to
pathogens via, for example, aerosols, splashes,
accidental inoculation.
• GMT are fundamental to laboratory safety.
• Specialized equipment may support good
laboratory practice but does not replace it.
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Personnel qualifications: proper training
For culture procedure:
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TB disease and transmission;
wearing and use of protective equipment and clothing;
handling of infectious materials;
laboratory design, including air flow conditions;
use of BSC, centrifuge (operation, identification of malfunctions,
maintenance);
preventing and coping with incidents;
good laboratory practice and good microbiological techniques;
organization of work flow procedures;
waste management;
importance of laboratory results for patient management;
importance of laboratory results for the national TB programme.
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Personal protective equipment
• Masks/respirators
• Gloves
• Gowns
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Personal protective equipment – masks
• Surgical masks prevent the
spread of microorganisms
from the wearer (protection
from exhalation).
• Masks do not provide
protection to the wearer
against inhaling small
infectious aerosols.
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Personal protective equipment –
respirators
N95/FFP2 respirators effectively filter out more than 95%
(98% for FFP3) of particles 0.3 µm or more in diameter.
Respirators have to be fitted to the face. Facial hair causes
the respirator to be ineffective.
N95/FFP2
FFP3
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Personal protective equipment –
respirators
Respirators may be worn in settings of high MDR-TB
and/or HIV prevalence, where staff may be HIV-infected
and highly susceptible to infectious aerosols.
Respirators must be available in all culture/DST labs for
coping with spillages outside the BSC.
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Personal protective equipment – gloves
• Gloves should always be worn while specimen
processing and handling TB cultures.
• Wearing gloves can give technicians a false sense of
safety.
• Frequent hand-washing and care in the handling of
contaminated materials are good laboratory practices.
• Gloves should be removed at every interruption of work
and should not be reused. Hands should be washed
after removal of gloves.
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Personal protective equipment – gowns
• Gowns must always be
worn inside the laboratory
(but never outside) and
should be changed
regularly, at least weekly.
• Gowns should preferably
be fully buttoned, longsleeved with narrow cuffs,
and back-opening.
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Removing PPE
Remove PPE in the following order:
1.Disposable gloves
1.Respirator/mask
1.Gown/coat/suit/overalls
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Personal protective equipment –
conclusion
• Hand-washing and GMT are acceptable
practices for most countries.
• Each country must evaluate the risks and
decide on the level of personal protection
equipment that is appropriate, given the
available resources.
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Disinfectants
• A disinfectant is a chemical or mixture of chemicals used to
kill microorganisms.
• Disinfectants are usually applied to surfaces or inanimate
objects.
• Disinfectants may be used for pre-decontamination
treatment, before autoclaving.
Because of their specific cell wall structure,
TB bacilli are resistant to most standard disinfectants,
e.g. quaternary ammonium compounds are ineffective.
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Disinfectants
The only adequate disinfectants against TB bacilli are:
• Phenol, 2–5%: very irritant to skin, use derivatives.
• Hypochlorite (bleach): corrosive to metals.
• Alcohol 70%, no residue, use on skin and work surfaces.
• Iodophores, 3–5%, iodine plus inert polymer.
• Glutaraldehyde, requires an activator, highly irritant to
skin.
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Disinfectants
• Diluted solutions should be prepared daily.
• Stock solutions should be stored according to
manufacturer's recommendations.
• Use commercial solutions for “dirty or worst possible
situations”.
• Follow national chemical safety guidelines.
[to be adjusted to local policy]
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Waste disposal
• All infected materials, including closed specimen
containers, should be placed in the BSC in
autoclavable bags.
• All cultures and related materials should be
autoclaved.
• All material handled in the BSC should be
considered as infected.
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Incident: spill
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Spill in the BSC – what to do
• Cover the spill with absorbent paper towel.
• Pour over disinfectant and leave for at least 2 hours.
• Discard absorbent tissue and all clean-up material in an
autoclavable bag and autoclave.
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Spill outside the BSC
• EVACUATE the room and stay outside with the door
closed for at least 2 hours.
• Using appropriate respiratory protection devices, return
to the accident area to clean the spill.
• Cover the spill with paper towel and pour over
disinfectant. Leave for at least 2 hours.
• Once disinfection is complete, discard all waste into
suitable waste containers and autoclave.
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Spill outside the BSC
Emergency spill kit should be available in all
culture/DST laboratories, with:
•all personal protective equipment (overalls, overshoes, gloves, respirators);
•Paper towels;
•large quantities of disinfectant.
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Chemical safety
• Alcohols are flammable: avoid flame
• Phenol is corrosive:
– avoid direct contact with the skin or mucous
membranes;
– limit exposure to phenolic fumes.
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Chemical safety: handling acids
ALWAYS ADD ACID TO WATER
NEVER ADD WATER TO ACID
WATER
ACID
WATER
ACID
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SAFETY FIRST!
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True and false exercise
1. Surgical masks protect you from TB infection.
2. Remove personal protective equipment in the
following order:
respirator/mask
disposable gloves
gowns/coats/suits/overalls.
3. In case of spills outside the BSC, you should
evacuate the room and stay outside with the door
closed for at least 30 minutes.
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Module review: take-home messages
 Never smoke, eat, or drink in the laboratory.
 Wash your hands frequently with soap and water
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before and after performing any procedures.
Avoid hazards in a TB laboratory by paying
careful attention to safety procedures.
Always work carefully and in a safe manner
following good microbiological technique.
The additional protection conferred by
respirators must be assessed for each setting.
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Self-assessment
• How is TB transmitted from person to person?
• What are the sources of infectious aerosols in a laboratory?
• What is the critical equipment for safe culture and DST?
• What personal protective equipment is recommended for TB
laboratory activities?
• What are the most efficient disinfectants in TB laboratories?
• What procedures should be followed when coping with
accidents?
• What precautions should be taken when handling acids?
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